Scientific basis for the project

Students will knock out a Saccharomyces cerevisiae gene involved in methionine biosynthesis (MET15) and replace it with the gene for nourseothricin resistance (NatMX). Successful gene editing will result in a yeast that will be unable to grow in the absence of methionine and resistant to the antibiotic nourseothricin. Students screen yeast for these phenotypes to determine the success of gene editing.

Brief summary of each session

The three sessions outlined below are all lab-based. The first two sessions involve plating out yeast and there needs to be enough time for yeast to grow before the next session (this should be at least 72 hours but growth can take longer after session 1).

The first session can be long! We recommend planning the pre-lab work to be delivered in advance (we do it asynchronously).

This used to be a longer series of practicals, which means that various experiments could be added back into the series if more sessions were desired or required. For example, linearisation of the repair template; PCR genotyping of transformants.

Each session also has a non-lab activity (paper or computer-based) that involves planning, design, or analysis. These are intended to supplement the students’ understanding of the practicals but they also help fill the long incubation steps, which can be annoying for students. However, these aspects could be delivered in a computer room as a separate session.

Session 1

A leu2- strain of yeast (K699) is transformed with two plasmids:

1. CRISPR plasmid: A plasmid expressing Cas9 and a single guide RNA (sgRNA) targeting the MET15 gene. This plasmid has a selectable marker (the LEU2 gene).
2. Repair plasmid: A plasmid that encodes the desired repair template. This plasmid has been previously linearised with restriction enzymes (this makes it a better substrate for homology-direct repair).

Following transformation, yeast are plated onto -leu plates, which selects for the presence of the CRISPR plasmid. Students also spend some time researching the plasmids so that they can identify and understand the role of the plasmid features.

Session 2

Colonies are picked from the plates and used to set up two screens:

1. Methionine auxotrophy. Colonies are picked and resuspended. The resuspension is serially diluted and plated onto -met plates. A positive and negative control are included for comparison.
2. Nourseothricin-resistance. The same colonies are streaked onto plates containing nourseothricin to test for resistance.

Students also design a single guide RNA.

Session 3

Plates from session 2 are analysed and the phenotypes of each colony recorded. From this, the genotype and outcome of gene editing are deduced. Students design a PCR strategy that would allow them to genotype their colonies and identify successful gene editing events.

Learning objectives

These practicals aim to:

1. Demonstrate how CRISPR/Cas9 gene editing technology can be used to modify the  genome of Saccharomyces cerevisiae.
2. Provide training in designing single guide RNAs for gene editing.
3. Demonstrate how online databases can be used when designing experiments.
4. Provide training in primer design for DNA analysis.

By the end of these practical sessions, you will have carried out the following experimental techniques:

1. Transformed DNA into S. cerevisiae.
2. Screened yeast transformants for resistance to nourseothricin.
3. Screened yeast transformants for methionine auxotrophy.
4. Designed a single guide RNA (sgRNA).
5. Designed PCR screening primers.
6. Kept an accurate and thorough record of your work in a lab book.

If you decide to use this practical, please let us know about it by filling in this short form – it’s not a requirement but we’d love to hear how our ideas are being used!